Lysozyme salts

ABSTRACT

Lysozyme glutamate or aspartate having anti-inflammatory and antimicroorganism action, and having much more stability to heat than lysozyme itself and having long shelf life, which is prepared from human placentae by extracting lysozyme fraction from frozen human placentae with NaCl treating the fraction with a cation exchange resin, eluting, controlling pH in two steps, i.e., 2.0 -4.0 and then 6.0- 9.0, centrifuging, desalting, adding glutamic or aspartic acid to the supernatant solution, heating at 60* C. for 10 hours and adding sulfated polymer to recover the desired lysozyme salts.

United States Patent [72] lnventors Tadakazu Suyama Hirakata-shl;Ken-ichl lzaka, Kadeom-shi; Hiroshi Shirakawa, Yamatotakada-shi, all ofJapan [211 App]. No. 788,648 V [22] Filed Dec. 30,1968 [45] PatentedDec. 14, 1971 [73] Assignee The Green Cross Corporation Osaka, Japan[32] Priority July 16, 1968 [3 3 Japan [3 l 43/50121 [54] LYSOZYME SALTS6 Claims, No Drawings [52] 0.8. CI 195/63, 195/68 [51] lnt.Cl Cl2k 1/00,C07g 7/02 [50] Field of Search 195/62, 63,

[56] References Cited UNITED STATES PATENTS 2,806,815 9/1957 Singhet etal. l95/63 OTHER REFERENCES Petit et al., Chemical Abstracts. Vol. 59Sept. 1963 (54l6e).

Primary Examiner-Lionel M. Shapiro Assistant ExaminerD. M. NaffAttorney-Christen, Sabol & OBrien ABSTRACT: Lysozyme glutamate oraspartate having anti-inflammatory and antimicroorganism action, andhaving much more stability to heat than lysozyme itself and having longshelf life, which is prepared from human placentae by extractinglysozyme fraction from frozen human placentae with NaCl treating thefraction with a cation exchange resin, eluting, controlling pH in twosteps, i.e., 2.0 -4.0 and then 6.09.0, centrifuging, desalting, addingglutamic or aspartic acid to the supernatant solution, heating at 60 C.for l0 hours and adding sulfated polymer to recover the desired lysozymesalts.

LYSOZYME SALTS The present invention relates to a process for preparinglysozyme salts. More particularly, the present invention relates to aprocess for preparing lysozyme glutamate and lysozyme aspartate fromhuman placentae.

Lysozyme is known as a mucopolysaccharidase having strong bacteriolyticaction. It has an anti-inflammatory action to mucous membranes and is,therefore, useful in the treatment of inflammatory diseases. Thelysozyme salts have substantially the same action as those of lysozyme.According to J. P. Jolles [Bull. Soc. Chem. biol. 45, 21 l, 1963] whodiscloses purification and analysis of lysozyme extracted from humanplacenta, to minced placenta is added the same volume of cooleddistilled water, pH is controlled to 4.5 with acetic acid, precipitateis centrifugalized, the solution is heated at 100 C. for l min. see.under pH 4.5 and then rapidly cooled in ice water, the solution isstanding overnight at 4 C. and centrifuged, the supernatant is treatedwith Amberlite CG-SO (200-400 mesh, pH 6.5, treated with 0.2 MNaphosphate buffer), decantated and further washed with 0.2 M aNat-phosphate buffer solution, lysozyme is eluated at pH 6.5 with 0.8 Ma Na-phosphate buffer solution and dialyzed at 4 C., pH is controlled to4.5 with acetic acid to centrifuge in order to obtain crude lysozyme,said lysozyme is treated with carboxymethylcellulose at pH 5.5 andwashed with 0.03 M a Na-phosphate buffer solution at pH 5.5, and thengradient elutions are effected at pH 5.5 to 7.2 with 0.03 M aNa-phosphate buffer solution and then at pH 7.2 with 0.1 M aNa-phosphate bufier solution to obtain purified lysozyme. French Pat.No. 1,444,176 discloses preparation of lysozyme ascorbate from egg whiteby adding NaCl thereto, precipitation impurities at pH 9.8, standing thesolution at pH 9.8 to give a precipitate of lysozyme, dissolving theprecipitate in H O, adding HCl to pH 4.5 in order to separateprecipitate, controlling pH finally to 9.8 with NaCl and Na Co in orderto produce precipitate of lysozyme, repeating the dissolution and theprecipitation as above of the precipitate to obtain purified lysozyme,adding water and then ascorbic acid to a paste of lysozyme, filtering anopalescent solution thus obtained to obtain a solution of lysozymeascorbate and treating the solution under CO to collect crystals of theascorbate.

An object of the present invention is to provide a method for preparingthe lysozyme salts from the human placentae. Another object of thepresent invention is to prepare lysozyme glutamate and lysozymeaspartate from the human placentae in a commercial scale, which are safefor venous injection and free from pyrogen.

Lysozyme prepared from the human placentae is without fear ofcontamination by heterogenous proteins and is completely absent fromanaphlaxylike side effects caused by the heterogeneous proteins. Thehuman placentae are, therefore, suitable for the source of lysozyme.However, lysozyme derived from the human sources is heat-labile andloses 80-90 percent of activity even heating at 60 C. for 10 hours.

The inventors have found that lysozyme can be isolated in pure form andmade heat-stable by converting it into the corresponding salts bytreatment with glutamic acid or aspartic acid. According to the presentinvention, lysozyme extracted from human placentae is reacted withglutamic acid or aspartic acid at 60 C. for 10 hours to produce lysozymesalts which are stable to heat. Sulfated polymer such as chondroitinsulfuric acid and dextran sulfate is added to the salts to precipitatecontaminating proteins, with the result that purified lysozyme which issuitable for injection is obtained.

According to the present invention, a solution of sodium chloride isadded to fresh or freshly preserved human placentae to extract the fracgonsco taliing lyspayme, lysozyme is collected on a cation exchangeresin equilibrated with a 0.05 M phosphate butter solution (pH 6.0-7.5),lysozyme is eluted from the cation exchange resin with a dilutedalkaline solution (pH 9.5-1 1.0) such as NaOH, KOH and (NH,) HPO,,neutral salts such as Na so MgSO, or such organic solvents as ethanoland the like are added thereto to precipitate the fraction containinglysozyme, a diluted acidic solution such as HCl, acetic acid is added tothe fraction to control pH at 2.0 to 4.0, the pH is controlled again at6.0 to 9.0 with KOH or NaOH, centrifugal force is applied to removehemoproteins, the supernatant lysozyme solution is desalted, pH iscontrolled at 7.0-8.0, glutamic acid or aspartic acid is added theretoand the mixture is heated at 60 C. for 10 hours to form the glutamate oraspartate of lysozyme, sulfated polymer is added to the products toprecipitate contaminating proteins, the precipitate is centrifugalized,and the supernatant fraction is sterilized.

The reaction of lysozyme with amino acids having affinity to lysozymeproduces the heat-stable products, the activity of which is notdestroyed during the reaction. The product salts can be preserved atroom temperature for an extended period of time. The infection sourcessuch as hepatitis virus and the like contained in the raw materials arekilled by the heat-treatment and the contaminating proteins are alsoremoved by addition of the sulfate polymers. The present salts arecompletely safe even in the intravenous injections and sterileinjectable.

The phosphate buffer solution should have pH value of 0.6 to 7.0. The pHvalue mentioned above is optimum in selectively adsorbing lysozyme onthe cation exchange resin and removing albumin and a part ofcontaminating protein such as hemoproteins. The cation exchange resin ispreferably carboxymethylcellulose and carboxymethyldextran. Sincelysozyme is a strongly basic protein it is selectively and stronglyadsorbed on the cation exchange resin at pH 6.0-7.0. It is hardly elutedat neutral pH, but efficiently eluted by an alkaline solution having pHvalue of 9.5 to H0 Ammonium sulfate or other neutral salts, or organicsolvents such as ethanol, acetone and the like are preferable toprecipitate the fraction containing lysozyme from the eluate.

Lysozyme come from human placentae contains a large amount ofhemoprotein. Almost all hemoproteins precipitate by adjusting pH in twosteps, i.e., firstly to 2.0-4.0 and subsequently to 6.09.0. Lysozymeremaining in the supernatant has the specific activity 5 to 6 times ashigh as the activity of the raw materials. However, it is still low inactivity to be used as an injectable medicine.

In order to increase the specific activity, desalination of the lysozymecontaining supernatant thus obtained is effected by gel filtration onthe cross-linked dextran having approximate exclusion limit (molecularweight) of 5,000 and particle size of I00 to 300. Glutamic acid oraspartic acid is added to the desalted solution at the pH value of 7.0to 8.0. The reaction of lysozyme with these amino acids is effected at60 C. for 10 hours and lysozyme glutamate or lysozyme aspartate isproduced by the reaction. After completion of the reaction, the reactionmixture is cooled to 10 to 20 C. and the sulfated polymer such aschondroitin sulfate and dextran sulfate is added thereto in theproportion of 0.1 to 1.0 percent to precipitate the contaminatingproteins. After removal of the contaminating proteins by centrifugation,the lysozyme fraction in the supernatant is sterilized and filtered togive sterile injectable lysozyme. The specific activity of lysozyme isincreased to 25 times as high as that of the raw materials by thepurification treatment.

Properties of the present aspartate and glutamate are shown in thefollowing table comparing with lysozymes from human placenta and fromegg white.

Lysozyme aspartate Lysozyme glutamate Lysozyme (from human (from humanplacenta) (from human placenta) placenta) Lysozyme (from egg white)Pharmacologic Bacterlolysls, antl- Bacteriolysis, anti- Bacterlolysls,antiactlvity. anaphylaxls, anti-virus, anaph laxis, anti-virus,auaphylaxis, anti-virus,

potentiatlon of antipotent atlon of antipotentiatlon of anti- Lysozymeaspartate (from human placenta) Lysozyme glutamate (from human placenta)Lysozyme (from human placenta) Lysozyme (from egg white) Active against:M.

Active against: M. lysodcikticus, B. lysodcz'kticus, B.

megatherium, B. subtilzs, Sarcina lutea,

subtzlts. Sarcz'na flava. Achro'mbacter fischeri,

Thermophilz'c bacterium, Staphylococcus aurens, Proteus rulgaris. 2.01:8.0 2.0 to 8.0. 25 to 37 2.5 to 55.

70 to 80% is lost: at 60 0., Not at 100 0., 45 min.

lllihrs; or 100 (3.,

The present invention is illustrated by a practical example.

The placenta delivered is immediately frozen in a freezer to preventputrefaction and to keep fresh. These frozen placentae are collectedfrom hospitals and maternity hospitals. Each 500 placentae is crushedroughly in an ice crusher and cut into small pieces by a rneatcutter. Toabout 270 kg. of thus finely cut placentae is added 500 l. of a 0.05 Msodium chloride solution and the mixture is agitated for 30 minutes toextract the lysozyme fraction. The mixture is centrifuged to separatethe extract. To the extract is added 1,500 g. of carboxymethylcelluloseequillibrated with a 0.05 M phosphate buffer solution having pH value of6.5. The mixture is agitated for 60 minutes and is subsequently allowedto stand. The supernatant is removed and the precipitates are collectedby either filtration or centrifugation. The collected precipitates arewashed with a 0.05 M phosphate buffer solution having pH value of 6.5until the washings become colorless and clear. The precipitates aresuspended in a 0.5 M aqueous sodium chloride solution and pH value ofthe solution is adjusted to 10.0 with addition of a 5 N aqueous sodiumhydroxide solution. The solution is either filtered or centrifuged toextract lysozyme.

Ammonium sulfate is added to the extract with agitation until the finalconcentration thereof reaches 30 percent by weight based on the totalmixture. The mixture is agitated for 60 minutes to precipitate globulinand a part of hemoproteins. The precipitates are removed bycentrifugation and ammonium sulfate is again added to the supernatantuntil the final concentration thereof reaches 60 percent by weight basedupon the total mixture and agitation is continued for 3 hours toprecipitate lysozyme. The lysozyme is dissolved in distilled water andthe solution is filtered. The filtrate is a concentrated solution oflysozyme. The pH of the solution is adjusted to 2.5 with 5 Nhydrochloric acid and the solution is allowed to stand for minutes andthe pH thereof is again adjusted to 7.5 and allowed to stand for 15minutes. The majority of the hemoproteins is removed by centrifugation.

The supernatant is desalted by use of SipilagQt G 25.

Aspartic acid is added tot he 'sfiutiiifiin the rate of 10 mg. per ml.after adjustment of the pH thereof to 7.5. The mixture is reacted at 60C. for l0 hours to form lysozyme aspartate. The reaction mixture issubsequently cooled to 10 to 20 C. and chondroitin sulfate is addedthereto in the proportion of 0.3 percent by weight to precipitate thecontaminating proteins. The precipitate is removed by centrifugation.The lysozyme fraction in the supernatant is sterilized by filteration toobtain about 62,000 mg. of the desired sterile product. The reactionconditions are almost the same in the reaction with glutamic acid and inuse of dextran sulfate as in that with aspartic acid and chondroitinsulfate.

What is claimed is:

l. A process for preparation of lysozyme salts which comprisesextracting lysozyme from human placentae preserved in a frozen stateimmediately after deliveries, collecting the lysozyme from the extracton a cation exchange resin, washing the adsorbed lysozyme and theneluting the lysozyme with a dilute alkali solution, initiallycontrolling the pH of the eluate to 2.0 to 4.0 and subsequently to 6.0to 9.0, centrifuging the solution to remove hemoproteins, desalting thesupernatant solution, adding thereto an amino acid selected from thegroup consisting of glutarnic and aspartic acids in a finalconcentration of about 10 mg. per ml., heating the mixture at 60 C. for10 hours, adding a sulfated polymer to the reaction mixture to removeprotein impurities and recovering the lysozyme salts.

2. The process according to claim 1 wherein the amino acid is glutamicacid.

3. A thennally stable lysozyme glutamate prepared according to theprocess of claim 2.

4. The process according to claim 1 wherein the amino acid is asparticacid.

5. A thermally stable lysozyme aspartate prepared accord ing to theprocess of claim 4.

6. The process according to claim 1 wherein the extraction from humanplacentae is effected with sodium chloride.

l l l k

2. The process according to claim 1 wherein the amino acid is glutamicacid.
 3. A thermally stable lysozyme glutamate prepared according to theprocess of claim
 2. 4. The process according to claim 1 wherein theamino acid is aspartic acid.
 5. A thermally stable lysozyme aspartateprepared according to the process of claim
 4. 6. The process accordingto claim 1 wherein the extraction from human placentae is effected withsodium chloride.